Effect of Calcium-Infiltrated Hydroxyapatite Scaffolds on the Hematopoietic Fate of Human Umbilical Vein Endothelial Cells.

Foamed hydroxyapatite offers a three-dimensional scaffold for the development of bone constructs, mimicking perfectly the in vivo bone structure. In vivo, calcium release at the surface is assumed to provide a locally increased gradient supporting the maintenance of the hematopoietic stem cells niche. We fabricated hydroxyapatite scaffolds with high surface calcium concentration by infiltration, and used human umbilical vein endothelial cells (HUVECs) as a model to study the effects on hematopoietic lineage direction. HUVECs are umbilical vein-derived and thus possess progenitor characteristics, with a prospective potential to give rise to hematopoietic lineages. HUVECs were cultured for long term on three-dimensional porous hydroxyapatite scaffolds, which were either infiltrated biphasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analyzed for typical hematopoietic and endothelial gene expressions, surface markers by flow cytometry, and hematopoietic potential using colony-forming unit assays. Our results indicate that the biphasic foams promoted a hematopoietic lineage direction of HUVECs, suggesting an improved in vivo-like scaffold for hematopoietic bone tissue engineering.

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis.

The accumulation of bacteria in surface-attached biofilms can be detrimental to human health, dental hygiene, and many industrial processes. Natural biofilms are soft and often transparent, and they have heterogeneous biological composition and structure over micro- and macroscales. As a result, it is challenging to quantify the spatial distribution and overall intensity of biofilms. In this work, a new method was developed to enhance the visibility and quantification of bacterial biofilms. First, broad-spectrum biomolecular staining was used to enhance the visibility of the cells, nucleic acids, and proteins that make up biofilms. Then, an image analysis algorithm was developed to objectively and quantitatively measure biofilm accumulation from digital photographs and results were compared to independent measurements of cell density. This new method was used to quantify the growth intensity of Pseudomonas putida biofilms as they grew over time. This method is simple and fast, and can quantify biofilm growth over a large area with approximately the same precision as the more laborious cell counting method. Stained and processed images facilitate assessment of spatial heterogeneity of a biofilm across a surface. This new approach to biofilm analysis could be applied in studies of natural, industrial, and environmental biofilms.

Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture.

Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain.

The degradation behavior of calcium-rich hydroxyapatite foams in vitro.

Hydroxyapatite (HA) is a well-known regenerative biomaterial. However, the slow degradation rate of HA is still an obstacle in clinical applications. In this study, we concentrated on investigating the degradation behavior of the calcium-rich HA foams, which had a demonstrated effect on blood differentiation in previous studies. The HA foams were processed by an emulsion method and were infiltrated with calcium nitrate to create a calcium carbonate second phase, heterogeneously distributed on and under the surface of the foam. During the 28-day solubility test, the calcium carbonate phase contributed to enhanced Ca2+ ion release into the saline compared to phase pure HA foams. Both types of foams were biocompatible as demonstrated by human endothelial cell culture on their surface. The release of calcium ions, the degradation behavior, and the endothelial cell differentiation behavior suggest this biphasic ceramic is a candidate for bone marrow in vitro culture and a possible bone substitute material.

Tissue Engineering and Regenerative Medicine Therapies for Cell Senescence in Bone and Cartilage.

The bone repair and substitute have been developed for decades with the bone defect repair applied successfully in clinical. However, implant complications may occur in more challenging situations, bone cell senescence and osteoporosis (OP). Due to certain microenvironment conditions, including hormonal, nutritional, and aging factors, the bone cell responses are regulated to alteration and end into cell senescence and OP. Thus, the bone tissue regeneration is limited with site morbidity increased, leading to bone graft failure. In such pathological state, bone grafts and substitutes are companied with OP therapies to improve bone tissue engineering and to enhance bone graft healing. Those substitutes with OP therapeutic-based applications have been becoming a growing field of interest in bone repair. Bone grafts, such as scaffolds with antiosteoporotic drugs releasing and implants with surface therapeutic modified, are studied to significantly increase low bone density as well as improve impaired bone regeneration. In this review, we discuss a thorough understanding in bone remodeling process, bone cell senescence, and mechanisms of OA and OP. Based on the understanding, the review concentrates on the treatments to the OP and the bone grafts applied in the bone senescence model and provides a future direction in clinical trial. Impact Statement This review discusses the tissue engineering and regenerative medicine therapies for aging-related bone diseases, which have been highly concerned in aging society. These therapies can improve the efficacy of bone tissue regeneration, enhance the quality of bone repair, and provide new insights in the field of bone clinical treatments and aging regeneration.

Calcium-Infiltrated Biphasic Hydroxyapatite Scaffolds for Human Hematopoietic Stem Cell Culture.

Long-term in vitro expansion of hematopoietic stem cells (HSCs), while maintaining their functionality and multilineage differentiation potential, is still challenging. In this study, three-dimensional (3D) high-porosity hydroxyapatite (HA) foams have been designed to closely mimic the chemistry and physical structure of cancellous bone. Furthermore, calcium oxide was distributed in the HA ceramics to provide surface calcium ion release, hypothesizing that a local surface calcium gradient supports HSC localization and maintenance. Primary human HSCs and osteoblasts were cocultured for 6 weeks. Controls were cultured in two-dimensional dishes, while scaffold cultures were performed with calcium nitrate-infiltrated HA scaffolds and untreated HA scaffolds. Cells were analyzed for surface markers by flow cytometry, metabolic activity, and hematopoietic multilineage differentiation potential. The release of calcium into culture medium was also determined. The implementation of HA scaffolds had a positive effect on erythrocyte colony formation capacity of HSCs, with an increased osteoblast fraction observed when compared to control cultures without scaffolds. The presentation of scaffolds did not affect metabolic turnover when compared to control cultures. In conclusion, 3D open-porous HA scaffolds provide a bone-like structure and enable the long-term maintenance of primary HSCs.

A microstructural study of the degradation and calcium release from hydroxyapatite-calcium oxide ceramics made by infiltration.

Hydroxyapatite pellets, partially densified in a low-temperature heat treatment, were infiltrated with calcium nitrate solution followed by in-situ precipitation of Ca(OH)2 and CaCO3. The infiltrated bodies were then densified to high relative density and the calcium carbonate transformed to calcium oxide during sintering and resulted in biphasic hydroxyapatite-CaO ceramics. This work investigated the influence of the infiltration on surface morphology, weight change, and microstructural-level degradation caused by exposure to saline at pH=7.4 and a temperature of 20°C. The CaO rendered the materials more susceptible to degradation, and released calcium into the saline faster than single phase, calcium deficient hydroxyapatite (HA) that were used as a control. In consequence, these ceramics could be used to release calcium into the culture microenvironments of bone tissue or bone marrow cells next to a scaffold surface.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells.

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

Induction of Hepatic and Endothelial Differentiation by Perfusion in a Three-Dimensional Cell Culture Model of Human Fetal Liver.

The development of functional engineered tissue constructs depends on high cell densities and appropriate vascularization. In this study we implemented a four-compartment three-dimensional perfusion bioreactor culture model for studying the effects of medium perfusion on endothelial, hepatic, and hematopoietic cell populations of primary human fetal liver in an in vivo-like environment. Human fetal liver cells were cultured in bioreactors configured to provide either perfusion or diffusion conditions. Metabolic activities of the cultures were monitored daily by measuring glucose consumption and lactate production. Cell viability during culture was analyzed by lactate dehydrogenase activity. Hepatic functionality was determined by the release of albumin and alpha-fetoprotein (AFP) in culture medium samples. After 4 days of culture, cells were analyzed for the expression of a variety of endothelial, hepatic, and hematopoietic genes, as well as the surface marker expression of CD31 and CD34 in flow cytometry. We found that medium perfusion increased the gene expression of endothelial markers such as CD31, von Willebrand factor (vWF), CD140b, CD309, and CD144 while decreasing the gene expression of the erythrocyte-surface marker CD235a. Hepatic differentiation was promoted under perfusion conditions as demonstrated by lower AFP and higher albumin secretion compared with cultures not exposed to medium perfusion. Additionally, cultures exposed to medium perfusion gave higher rates of glucose consumption and lactate production, indicating increased metabolic activity. In conclusion, high-density bioreactors configured to provide constant medium perfusion significantly induced hepatic and endothelial cell differentiation and provided improved conditions for the culture of human fetal liver cells compared with cultures without perfusion.

Antimycobacterial efficacy of silver nanoparticles as deposited on porous membrane filters.

Environmental mycobacteria pose a significant health burden. Non-tuberculous mycobacteria infections have been traced to water treatment networks, where mycobacterial biofilms are ubiquitous. Filters that remove potential pathogens have significant medical applications. The purpose of this study is to demonstrate that an antibacterial silver nanoparticle (AgNP) coating can prevent colonization and growth of a mycobacterial biofilm on a filter material. The antibacterial efficacy of commercially available AgNPs was measured against Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium marinum after 48 h in liquid culture. Nanoparticles were deposited on micro-porous track etched polycarbonate membranes. The growth of biofilms on the membranes was observed by microscopy and counting colony forming units. M. smegmatis was most susceptible to AgNPs, with a 98.7% reduction at 100 µM AgNP concentration. M. avium was reduced by 97.3% at 539 µM AgNP after 48 h. Deposited nanoparticles inhibited colonization and growth for both M. smegmatis and M. avium on the membrane surface. Similar to the liquid culture, M. avium (84.2% survival) was more resistant than M. smegmatis (0.03% survival).